Orgotein production using a buffer solution containing divalent metal salts

ABSTRACT

THE ISOLATION OF ORGOTEIN FROM AN AQUEOUS SOLUTION OF A MIXTURE OF PROTEINS COMPRISING IT IS FACILITATED BY CONDUCTING THE ISOLATION IN AN AQUEOUS SOLUTION CONTAINING A DIVALENT METAL HAVING AN IONIC STRENGTH OF 0.60 TO 1.00 A., PREFERABLY COPPER, ZINC OR BOTH.

United States Patent 3,832,338 ORGOTEIN PRODUCTION USING A BUFFEREXSTION CONTAINING DIVALENT IVIETAL Wolfgang Huber, San Francisco, andThomas L. Schulte, Wood-side, Calif., assiguors to Diagnostic Data,Inc., Mountain View, Calif.

N 0 Drawing. Continuation-impart of application Ser. No. 237,507, Mar.23, 1972, now Patent No. 3,758,682, which is a continuation-in-part ofapplication Ser. No. 15,883, Mar. 2, 1970, which is acontinuation-in-part of application Ser. No. 3,538, Jan. 16, 1970, whichis a continuation-in-part of application Ser. No. 576,454, Aug. 31,1966, which in turn is a continuation-in-part of application Ser. No.494,048, Oct. 8, 1965, all now abandoned. This application Feb. 7, 1973,Ser. No. 330,401 The portion of the term of the patent subsequent to May18, 1988, has been disclaimed Int. Cl. A61k 17/00; C07g 7/04 US. Cl.260-113 Claims ABSTRACT OF THE DISCLOSURE The isolation of orgotein froman aqueous solution of a mixture of proteins comprising it isfacilitated by conducting the isolation in an aqueous solutioncontaining a divalent metal having an ionic strength of 0.60 to 1.00 A.,preferably copper, zinc or both.

BACKGROUND OF THE INVENTION This invention relates to a process for theisolation and purification of orgotein. This is a continuation-in-partof Application Ser. No. 237,507, now US. 3,758,682, filed Mar. 23, 1972as a continuation-in-part of Application Ser. No. 3,538, now abandoned,filed Jan. 16, 1970, as a continuation-in-part of Application Ser. No.15,883, now abandoned, filed Mar. 2, 1970, as a continuation-in-part ofApplication Ser. No. 576,454, filed Aug. 31, 1966, now abandoned, as acontinuation-in-part of Application Ser. No. 494,048, filed Oct. 8,1965, now abandoned.

Orgotein is the non-proprietary name assigned by the United StatesAdopted Name Council to members of a family of water-soluble proteincongeners in substantially pure, injectable form, i.e., substantiallyfree from other proteins which are admixed or associated therewith inthe sources thereof. In lyophilized form these proteins aregreenish-white powders soluble in water, saline, and butter solutionsand injectable without manifesting toxicity or the immunologic reactionstypical of foreign-body proteins. Their elemental, infrared,ultraviolet, spectrographic, optical rotary dispersion and otheranalyses are consistent with their metalloprotein chelate structure. Thenovel pharmaceutical compositions of this invention are useful forameliorating and mitigating in humans and other mammals as well as inother animals the adverse effects of inflammatory conditions, of stressconditions, including shock and toxemia, and of certain viral diseasesas evidenced by pharmacological and clinical evaluation. See J.A.M.A.,May 26, 1969, Vol. 208, No. 8; Huber et al. Abstracts Seventh AnnualMeeting of the Society of Toxicology, Washington, DC, March 1968; Carsonet al., Federation Proceedings, 29, 420 [978] (1970).

The metalloproteins of the compositions of this invention are members ofa family of protein congeners having a characteristic combination ofphysical, chemical, biological and pharmacodynamic properties. Each ofthese congeners is characterized physically by being the isolated,substantially pure form of a globular, buffer and Watersoluble proteinhaving a highly compact native conformation which, although heat labile,is stable to heating for several minutes at 65 C. when dissolved in abuffer solu- "ice tion containing a salt of a divalent metal having anionic radius of 0.60 to 1.00 A. and which on gel electrophoresis gives acharacteristic multiple-band pattern. Chemically, each is characterizedby containing all but 0-2 of the protein aminoacids, a small percentageof carbohydrate, no lipids, 0.1 to 1.0% metal content provided by one to5 gram atoms per mole of one or more chelated divalent metals having anionic radius of 0.60 to 1.00 A., and substantially no chelatedmonovalent metals or those that are cell poisons in the molecule.Pharmacodynamically, each of the congeners is characterized by being anon-toxic, only weakly immunogenic injectable protein Whosepharmacodynamic properties include anti-inflammatory activity.Immunological relatedness of an orgotein congener is sufficient toenable its antibodies prepared in the rabbit or other suitable animal torecognize as an antigen one or more other orgotein congeners and/ or forone or more of the antibodies to other orgotein congeners to recognizeit as an antigen, as evidenced, for instance, in gelimmunoelectrophoresis and/or gel immunodiffusion. Although some of thephysical and chemical properties and the type and degree ofpharmacodynamic efficacy of orgotein vary from congener to congener, allorgotein congeners possess the above combination of properties.

From recent literature data, it is now apparent that this family ofmetalloproteins includes the proteins previously isolated in variousstates of purity and given the names hemocuprein and hepatocuprein. Mann& Keilin, Proc. Royal. Soc. for Biol. Sci., 126, 303 (1939);cerebrocuprein, Porter & Ainsworth, J. Neurochem., 1, 260 (1957);erythrocuprein, Markowitz et al., J. Biol. Chem., 234, 40 (1959); andcytocuprein, Carrico & Deutsch, J. Biol. Chem., 244, 6087 (1969). Forother references, see Mohamed & Greenberg, J. Gen. Physiol. 37, 433(1954); Porter & Folch, Arch. Neurol. Psychiat. 77, 8 (1957); Porter &.Ainsworth, J. Neurochem., 5, 91 (1959); Krimmel et al., J. Biol. Chem.,234, 46 (1959); Wyman, Biochem. Biophys. Acta, 45, 387 (1960); Shieldset al., J. Clin. Inv., 40, 2007 (1961); Markowitz et al., Anal. Chem.,33, 1594 (1961); Porter et al., Arch. Biochem. Bioph., 105, 319 (1964);Stansell & Deutsch, J. Biol. Chem., 240, 4299 (1965); ibid, 240, 4306(1965); Stansell & Deutsch, Clin. Chem. Acta, 14, 598 (1966); McCord &Fridovich, J. Biol. Chem., 248, 5753 (1968); Hartz & Deutsch, J. Biol.Chem., 244, 4565 (1969); McCord & Fridovich, J. Biol. Chem., 248, 60561968); Carrico & Deutsch, ibid, 245, 723 (1970. These metalloproteinshave reported to possess very high superoxide dismutase activity. SeeMcCord & Fridovich, J. Biol. Chem., 244, 6049 (1969); Keele, McCord andFridovich, J. Biol. Chem., 245, 6176 (1970); ibid, 245, 2875 (1971).

Orgotein is at least partially in the form of a metal chelate, i.e., itcontains from 1 to 5, preferably about 4, gram atoms per mole of protein(GAPM) of one or more chelated divalent metals having an ionic radius of0.60 to 1.00 A. Preferably, the predominant metal is one having an ionicradius of 0.65 to 0.79 A., i.e., Co, Cu, Fe, Ge, Mg, Ni and Zn, morepreferably Cu or Zn. Above 4 GAPM, at least a portion of the metalcontent of the protein appears not to be in chelated form and is notrequired for the physiological activity of the protein to be manifestedat its maximum. The total metal content of most samples is about 0.1 to1% and in the most active samples, it is between 0.15 and 0.75%. Thedegree of pharmacological activity possessed by orgotein appears to beat least partially dependent upon the presence of one or morephysiologically essential divalent metal ions in the chelate. Therefore,preferably at least 65%, desirably at least and most preferably or moreof the metal content of the chelate is provided by a combination of oneor more of Ca, Cu, Fe, Mg, Co and Zn in divalent form, more preferablytwo or more of Cu, Mg,

C and Zn. For a list of ionic radii of metals, see Hall, Chemistry andPhysics, 44th Ed. pages 3507-8 (1962). In most of such samples, a metalhaving an ionic radius of 0.65 to 0.79 A., preferably Cu, Mg or Zn isthe predominant metal. By predominant metal is meant the chelating metalpresent in highest percentage in the protein chelate. Most orgoteincongeners are isolated containing about 2 GAPM each of Cu and Zn.

For a further description of orgotein, see the disclosure of SN. 237,507and the applications cited above parent thereto, which disclosure isincorporated by reference. See also Netherlands Pat. 66/14,177, BelgiumPat. 687,828 and British Pat. 1,160,151.

As is typical of processes for the isolation and/or purification of aprotein, orgotein is isolated and/or purified by one or more steps inwhich an aqueous solution of a mixture of proteins comprising orconsisting essentially of orgotein is fractionated to yield a fractionenriched in orgotein. Such fractionation steps typically employ one ormore of organic solvent or soluble salt fractionation and columnchromatography.

In the earliest of the prior filed parent applications cited above,orgotein is isolated in a multi-step process employing both organicsolvent and ammonium sulfate fractional precipitation, selective heatdenaturation and molecular sieve chromatography or gel electrophoresis.See also 3,687,927. 3,579,495 claims a process for the isolation oflysed red blood cells. 3,624,251 claims a postheat treatmentpurification of substantially pure orgotein. All of these processesinvolve the fractionation of an aqueous solution of a mixture ofproteins comprising orgotein, in an amount from as low as a few partsper million to substantially pure orgotein, to yield a protein fractionenriched in orgotein.

SUMMARY OF THE INVENTION The process of this invention is an improvementin processes for the production of orgotein which comprise at least onestep of fractionating an aqueous solution of a mixture of proteinscomprising orgotein to yield a fraction richer in orgotein than thestarting mixture of proteins, which improvement comprises conducting thefractionation step with a buffer solution containing dissolved thereinat least one salt of a divalent metal having an ionic radius of 0.60 to1.00 A.

DETAILED DISCUSSION In its preferred aspects, the process of thisinvention comprises one or more of the following:

(a) The fractionation is conducted in a plurality of steps;

(b) The fractionation comprises a heating step in which extraneousproteins are insolubilized;

(c) The divalent metal has an ionic radius of from 0.65 to 0.79 A.;

(d) The buffer solution contains dissolved therein a Cu++ salt; and

(e) The buffer solution contains dissolved therein a Zn' salt.

The isolation of orgotein from mixtures of proteins comprising it and/orthe purification of isolated orgotein is usually conducted in a buffersolution thereof. Typically, one or more fractionation steps areconducted in which a portion of the proteins of a mixture of buffersoluble proteins comprising orgotein is separated therefrom, e.g., byprecipitation, using heat, solvent or ammonium sulfate or other solublesalt; by column chromatography, e.g., using diethylaminoethylcelluloseor other weakly basic ion exchange resin.

According to the process of this invention, such fractionation steps areconducted in the presence of a divalent metal ion having an ionic radiusof 0.60 to 1.00 A., preferably 0.65 to 0.80 A., more preferably 0.65 to0.79 A. These ions can be provided by conducting the fractionationemploying an aqueous solution having dissolved therein a salt of theselected metal ion, e.g., Mg(OOCCH MgSO MgCl CaCI MnSO Cu(OOCCH Zn OOCCHZnSO etc., in at least 1 x 10* M concentration, preferably 0.005 to 0.20M, e.g., about 0.20 M, when the metal is Mg or Mn. The concentrationwhich should be used is dependent upon the divalent metal used.Magnesium, manganese, calcium and cobalt keep the protein in solutionabove about 0.2 M or below about 0.02 M concentration of the metal. Zincand copper bring about precipitation at 0.2 M. Therefore, these shouldbe used at lower concentration than the other metals, e.g., about 5 x10- M for copper and 55 x 10- M for zinc. Thus, the upper limit of saltconcentration is that which salts out the orgotein, the lower limitbeing that at which no significant protection is afforded the orgoteinprotein against loss of chelated metal.

To achieve substantial protection, the selected salt or salts ofdivalent metals should be present at a total concentration of about 24to 40 or more gram atoms per mole (GAPM) of orgotein. Thus, if naturalorgotein, which contains 2 GAPM each of chelated Cu++ and Zn++ in themolecule (about 0.4% by weight) is to be protected according to theprocess of this invention, the concentration of each metal should be12-20 or more GAPM of orgotein. In terms of concentration, this meansthat the aqueous solution employed in any fractionation of a mixture ofproteins comprising orgotein at usual concentrations should containabout 2.4 to 4.0% of each of Cu++ and Zn++ based on the orgoteinpresent, to ensure maximum protection against loss of these metals fromthe orgotein molecule and resultant loss of molecular conformation.Proportionately higher or lower amounts of other divalent metals ofhigher or lower atomic weight will be required.

In U.'S. 3,579,495, there is claimed an improvement in the process ofthis invention in which a heating step is conducted in a buffer solutioncontaining as the sole divalent metal ions, a mixture of Mg++, Cu++ andZn++ ions, in concentrations of 10 M, 10- M and 10- M, respectively.

Orgotein usually occurs as a mixed Cu++, Zn++ chelate containing about 2gram atoms per mole (GAPM) of each metal. Therefore, in its preferredaspect, the process of this invention is preferably conducted in abuffer solution containing dissolved therein a salt of one or both ofthese divalent metals. However, salts of other divalent metals having anionic radius of 0.60 to 1.00 A. are also effective in improving theisolation of orgotein. For example, a soluble salt of manganese is moreeffective than of other divalent metals in facilitating the extractionof orgotein from liver.

As stated above, orgotein ordinarily exists as a copperzinc mixedchelate. If a chelate is desired containing another divalent chelatingmetal having an ionic radius of 0.60 to 1.00 A. in addition to or insubstitution of one or more of these metals, such a chelate can beproduced by transchelating a chelate Whose predominant metal is onehaving an ionic radius of 0.65 to 0.79 A. or chelation of theapoprotein.

Transchelation techniques known in the art can be used, if appropriatelymodified to take into consideration the properties of orgotein, e.g., bydissolving orgotein in a buffer solution containing a water soluble saltof a divalent metal having an ionic radius of 0.65 to 0.79 A., with theratio of metal ion to protein being such as to retain the protein in theselected buffer solution as a metal chelate of such metal. The choice ofconditions is limited by the labile character of the orgotein protein inthe absence of chelating metals and at pH below 1 or above 1 1.

In addition to certain pH, the orgotein protein is sensitive to heat,the degree of sensitivity depending in part upon its degree of purityand concentration. When very impure, e.g., in the form of a lyophilizedmixture of all the soluble proteins from a natural source, the orgoteinis reasonably stable. The pure orgotein protein, in its metal chelatedform of this invention, is also stable, so that less care is required inhandling and storing' pure orgotein than substantially pure orgotein. V

In Application S.N. 291,320, filed Sept. 22, 1972, there is claimed animproved process for the isolation of orgotein from red blood cells inwhich at least the red blood cell portion of whole blood is heated at6080 C. to precipitate both the hemoglobin and carbonic anhydrase. InApplication Ser. No. 273,278, filed July 19, 1972, there is claimed theenzymatic removal of extraneous proteins from mixtures comprisingorgotein. 'In these processes, as in the processes of Applications Ser.No. 205,609 and 205,610, now US. 3,763,136 and 3,763,137, and US. Pats.3,579,495 and 3,624,251, the fractionation steps are preferablyconducted in an aqueous solution containing a salt of a divalent metalhaving an ionic radius from 0.60 to 1.00 A.

As stated above, the orgotein protein is sensitive to pH. Denaturationoccurs at pH below 1.0 and above 13. Therefore, solutions of the proteinshould be maintained at a pH from 4-10 to avoid reduced yields bypartial denaturation. The degree of solubility or insolubility under anygiven set of conditions is also dependent on the degree of purity andthe concentration of the desired protein.

Preferably the pH of the solution containing the divalent metal ions isnon-acidic, e.g., pH 7.0 to 8.5. When the fractionation step involvesprecipitation of less soluble proteins while retaining the orgotein insolution, the pH is preferably about 7-8.5. When the orgotein protein isprecipitated, the pH is preferably about 4-7.5, e.g., about 5.0.

Buffers which can be used include any conventional bulfered aqueoussolvent solution for proteins which provide the requisite pH, e.g., NH HPO NH HPO tris(hydroxymethyl)aminomethane, maleic acid-NaOH, citricacid-sodium citrate, acetic acid-sodium acetate, citric acid-(NH HPOsuccinic acid-NaOH, sodium acid maleate-NaOH, sodium cacodylate-HCl,boric acid-borax, etc. See G. Gomori, Methods in Enzymology, Vol. I,pages 138-146 (1955), especially buffers No. 5-8 and -18. Water adjustedto weakly alkaline pH and containing sufficient divalent metal ion canalso be used.

As stated above, the desired protein is much more stable, particularlyto heat, as a chelate of a divalent metal (Me++) having an ionic radiusof 0.60 to 1.00 A. It rapidly loses its stabilized compact configurationin the absence of adequate amounts of such chelating metals. Therefore,it is especially desirable that any heat purification step be conductedwith the process of this invention.

Starting Materials A 'wide variety of natural protein sources containtrace amounts of the precursor natural form of orgotein. Such sourcesinclude animal organs and tissue, e.g., liver, kidney, testes, pancreas,placenta, intestinal mucosa, thymus, lung, spleen and red blood cells ofthe rabbit, sheep, lamb, mules, horses, chickens, rats, monkeys, goat,guinea pig, dog, cat, swine, cows, steers, calves, humans, marineorganisms, e.g., whale, dolphin, sea lion, shark, swordfish, mussels,lobsters and oysters, vegetable sources rich in protein, e.g., seeds,wheat germ, whole rye, soya, kidney, lima and jackbeans, and mushrooms;also micro-organisms, e.g., fungi and bacteria, including yeasts, E.Coli, Streptomyces, penicillium and Saccharomyces. Preferred sources areanimal organs and red blood cells (RBC), preferably bovine.

The orgotein precursor is often extracted from the natural proteinsource along with proteins having enzyme activity, e.g., arginase andcarbonic anhydrase, etc. However, except for superoxide dismutaseactivity possessed by the Cu chelated congener, which is as high as anyreported in the scientific literature, orgotein itself does not exhibitgeneralized enzyme activity. Tests in over 30 different enzyme systems,utilizing a broad range of substrates, have failed to reveal anysignificant activity when the protein was used in lieu of the enzyme inthe respective assay systems. Included were several each of theoxidoreductases, transferases, hydrolases, proteases, lipases andisomerases. Only in the case of catalase, peroxidase and snake venomphosphodiesterase were traces of activity observed, i.e., less than 2%of that of the respective known enzymes run in parallel.

Known techniques for isolating such enzyme-containing fractions can beemployed for obtaining a starting protein-fraction containing enrichedamounts of the orgotein precursor if the isolation technique employs afreshly harvested source of protein and otherwise meets the requirementsfor non-destruction of the desired protein. See R. M. Morton in Methodsin Enzymology, Colowick and Kaplan Editors, Vol. I, pp. 25-51, AcademicPress, New York (1955).

To determine whether a mixture of soluble proteins contains proteinprecursors of orgotein, a divalent metal having an ionic radius of 0.60to 1.00 A. is added to a solution of the proteins and enough proteinimpurities are removed to permit the characteristic multi-band patterntypical for the orgotein protein in gel electrophoresis at low ionicstrength to be detected among the other proteins present. To do so, themixture of proteins to be assayed for orgotein protein content isdissolved at 0-5 C. in a buffer at pH between 1 and 13, e.g., 4-10,preferably about 7.5, which contains dissolved therein a salt of one ormore divalent metals. Any buffer-insoluble proteins are removed, e.g.,by filtration or centrifugation. The buffer-soluble proteins are thenprecipitated therefrom with a water-miscible solvent, e.g., acetone. Thebuffer soluble portion of the precipitated proteins will reveal on gelelectrophoresis on polyacrylamide or agarose at low ionic strength,Within its overall pattern the narrow, closely spaced multi-band patterntypical of orgotein. The details for running such electropherograms havebeen described above.

Thin film argarose electropherograms are particularly useful to followthe enrichment of the orgotein protein in the protein mixture. Sampleconcentrations of 1.0- mg./ml., run in 0.17 M tris-glycine buffer at pH8.45, 5 ma. and 200-300 v. for 30 minutes have proved useful for thispurpose. An advantage over disc gel electrophoresis as an analyticaltool is their ability to visualize both cathodically and anodicallymoving proteins as a result of the sample well being near the center ofthe plate.

Orgotein Isolation Procedure A typical isolation technique employing theprocess of this invention to obtain isolated substantially pure orgoteinfrom animal organs or tissue first removes insoluble materials, using anaqueous solution as a selective solvent for the soluble proteinscontaining the desired protein. Then materials more soluble than thedesired protein are removed employing one or more organic solvent and/orsalt precipitation steps in which a fraction of the proteins containingthe orgotein is precipitated and the more soluble material is retainedin the supernatant. Thereafter, undesired thermolabile soluble proteinsare insolubilized by a brief heating step which selectively denaturessuch proteins. Preparative electrophoresis, preferably gelelectrophoresis, or gel filtration can be used to remove residualundesirable, soluble proteins such as, for instance, albumin, therebyproviding an injectable protein product free of impurity-induced sidereactions.

To isolate the proteins comprising orgotein from natural sources, anygross particles of non-proteinaceous and fibrous insoluble proteinaceousmaterial from a freshly harvested source of protein can be removed in aconventional manner. As soon as possible, the freshly 7 harvestedmaterial should be chilled and kept chilled except as indicated herein.A temperature below 10 C. is desirable, preferably below C., e.g., asclose as practicable to the freezing point of the aqueous solutions usedin the isolation steps.

The first step in isolating orgotein from a protein source thereof bythe removal of insoluble proteins from a tissue source can be achievedby intimately mixing the finely divided protein mixture with an aqueoussolution, preferably a buffer solution, of a pH of 4-12, preferablyabout neutrality.

After separating the insoluble proteins from the buffer solution of thesoluble proteins, e.g., by filtration or centrifugation, separation ofthe undesired soluble proteins and any remaining non-proteins can beaccomplished at least in part, by selective precipitation. Much of theundesired highly soluble and less soluble proteins can be removed fromthe protein mixture by step-wise selective precipitation of the proteinsin the mixture from a buffer solution thereof, using organic orinorganic materials soluble in or miscible with, the buffer solution.For example, lipids, nucleic acids, nucleotides and other extraneousmaterials can be separated by adding suflicient cold acetone or otherwater miscible organic solvent to the buffer solution of the solubleproteins. Lipids and other acetone soluble impurities remain insolution. Most or all of the proteins, including the orgotein protein,are precipitated. On extraction of the precipitate with appropriate,buffer, the orgotein protein is dissolved while many of the otherundesired materials remain insoluble.

Any pigmentaceous material in the buffer solution ought also be removed.This can be accomplished by adding a water-soluble amine, preferablyheterocyclic, e.g., pyridine, piperidine, or other Water-miscibleorganic solvent or solvent mixture in which the pigmentaceous materialis insoluble, to a solution of the proteins in a buffer solution, e.g.,before the precipitation of proteins less soluble than the orgoteinprotein. Removal of the undesired less soluble proteins can follow, ifdesired, the separation of the precipitated pigmentaceous materials, byadding an inorganic salt or sufiicient additional organic solvent toselectively precipitate some proteinaceous material, leaving the desiredprotein and the more soluble, undesired proteins and any otherextraneous very soluble material in the supernatant. For example, thepigmentfree buffer solution of the soluble proteins initially can bebrought to 40-55% of saturation with ammonium sulfate or other organicor inorganic salt or by use of organic solvent at a concentration whichreduces the solubility of the protein mixture in the buffer, therebyselectively precipitating much of the undesired less soluble protein,which can be discarded. Organic materials which can be used toselectively precipitate undesired proteins include water miscible polarsolvents, e.g., lower aliphatic alcohols, such as ethanol and isopropylalcohol, and acetone, dioxane and tetrahydrofuran. For example, amixture of chloroform and ethanol can be used to precipitate thepigmentaceous material and then, after removing that pre cipitate, moreethanol is added until protein precipitation begins to occur. Ifdesired, the chloroform can first be removed under vacuum. Other organicsolvents, e.g., the lower-aliphatic alcohols, acetone, dioxane,tetrahydrofuran, can be used in this step.

Orgotein can be selectively precipitated from the buffer solution,leaving more soluble materials therein, by adding additional salt orsolvent thereto in the manner described for the removal of the lesssoluble proteins.

The selectivitiy and efliciency of these salt and solvent fractionationprecipitations of protein mixtures comprising orgotein are aifected bythe pH of the buffer solution, which should be maintained between 1 and12, preferably about 4-10, as well as by the ionic strength and thetechnique of addition.

An important step in the above-described fractionation process is aheating step in which the proteins more heat labile than orgotein aredenatured. This step is preferably conducted after buffer insolubleproteins and non-proteins, pigmentaceous material and organicsolvent/water soluble components have been removed. It is important thisstep takes place with the orgotein protein in the form of a divalentmetal chelate. Therefore, the heat treatment ought be conducted in abuffer solution of the protein mixture containing from 1X 10- to 2 10 vMor more, depending upon the metals used, of the ions of divalent metalhaving an ionic radius of 0.60 to 1.00 A.

In such a heating step, a buffer solution of the mixture of proteinscontaining the orgotein protein is heated at about -75 C. for a periodof time from a few seconds up to about 45 minutes, depending on theselected temperature.

The time and temperature employed in the heat treatment are inverselyproportional. At this point in the purification process, the desiredprotein i only briefly stable at temperatures above 75 C. Therefore,unless an instantaneous heating and chilling technique like flashpasteurization is employed, the mixture should not be heated above 75 C.Heating to temperatures below 50 C. usually is not satisfactory becausesome of the undesired heat labile proteins are fairly resistant todenaturation at such lower temperatures. At this point in thepurification process, the desired protein is stable at C. for at least15-30 minutes, at C. for about l025 minutes and at C. for about 1015minutes, thus permitting the use of conventional heating and coolingtechniques. Therefore, heating at about 55 C. for about one hour toabout C. for a few minutes, preferably about 60 to 65 C. for about 10 to20 minutes, is usually employed.

The amount of orgotein lost in the heating step is partially dependentupon the amount of protein impurities which are denatured and theorgotein concentration. However, the heating step can be conducted on aprotein mixture in which none, some, or substantially all of the proteinimpurities have been previously removed by other techniques.

In the final purification any significant remaining amounts ofextraneous proteins are removed to produce the isolated substantiallypure orgotein. Because the remaining proteins other than albumin typeare less apt to produce desirable responses upon injection, theirvirtually complete removal is less critical but nonetheless muchpreferred. Remaining extraneous proteins can be removed in a variety ofways, e.g., countercurrent extraction, gel filtration, paper or thinlayer chromatography, or selective elution from apatite and otherinorganic gels or ion exchange columns either singly or in combination.Gel electrophoresis or resin chromatography using a porous resin whichacts as a molecular sieve, e.g., crosslinked dextran, is preferred.Resin chromatography is most preferred for reasons of production economyand because larger amounts of protein can be processed at one time.

An albumin removal step is essential, when the protein source containsalbumin, because the other isolation steps usually employed in a processfor producing the desired protein product increase rather than decreasethe absolute albumin content of the purified protein. For example, thealbumin content of the total soluble protein fraction from bovine liveris 7.5%; bovine kidney, 8%; from porcine kidney, 10%; fron bovinespleen, oysters and mussels, 23%. In the fractionation steps describedhereinafter, albumin content of the concentrates rises to 223l%. Gelelectrophoresis or resin chromatography is effective in reducing thealbumin content of these concentrates to below 1%.

Thus, concentration without electrophoresis or resin chromatography of aprotein source containing significant amounts of albumin causes abuild-up of albumin which precludes its safe use as an injectablepharmaceutical agent and prevents it from manifesting usefulpharmacological activity. Free-falling curtain electrophoresis iscapable of removing much of this albumin. Gel electrophoresis and resinchromatography remove even more. An albumin removal step is not, ofcourse, required when albumin-free starting material, such as red bloodcells from many species, is used.

A commercially available electrophoresis unit which can be used forfree-falling curtain electrophoresis is the Brinkmann Model FF. Theseparating chamber in one such unit for instance is 50 centimeterssquare and 0.5 to 1 mm. in depth. The temperature is maintained as closeto 5 C. as possible. The unit permits the collection of up to 48fractions. In operation, the protein, dissolved in trismaleate-Me++buffer, pH 7.6, is applied continuously. Currents of about 1,000 voltsand -20 ma. are used. With properly pre-purified protein mixtures, thedesired protein chelate will be found in fractions 10-26 which arepooled, dialyzed and lyophilized. The construction and the operatingcharacteristic of this unit limit its capacity to about 500 mg. runs.The isolated protein is obtained in batches of about 100 mgs. which aresubsequently pooled. Using this method, albumin levels can be lowered toabout 510%. However, levels below 5% are not ordinarily achieved.

A more effective purification technique is the gel or zoneelectrophoretic purification described herein which uses a gelsupporting medium, e.g., polyacrylamide, agarose, starch, etc.Substantially complete removal of albumin and other extraneous proteinscan be achieved by this technique, by virtue of their different speedsof migration.

The preferred preparative gel electrophoresis media is polyacrylamide (5to 10%). Cellulose, cross-linked dextran (Sephadex, Pharmacia, Upsala,Sweden) and starch modifications (ethanolized, etc.), agar, sucrose-agarand other agar modifications are satisfactory but have the disadvantageof their gels being more fragile. For a description of the principles ofgel or zone electrophoresis, see Gel Electrophoresis, I F. Frederick,Editor, Annals N.Y. Academy Sci., 121, 305-650 (1964).

A production model developed for disc gel electrophoresis purificationhas a 5 to 7% polyacrylamide block 32 centimeters long, 10 centimeterswide and one centimeter deep held between jacketed top and bottom platesmade from clear plastic. The dimensions of the block are such thatcooling is very efficient and the small depth assures rapid temperatureequilibrium between center and surfaces. Cooling is provided by arefrigerated circulating system employing ethylene glycol-water.Operation is carried out at 600-1000 volts and 200-500 ma. Thesecurrents together with the very efficient cooling make it possible tohandle 1-5 g. quantities of starting protein during a developing processof 2-10 hours. The material is applied to a starting trough as a highlyconcentrated solution in tris-maleate-Me++ or similar buffer at pH 7.4.At appropriate times of development, buifer is passed through the gel atright angles to the direction of electrophoretic flow to elute theprotein. Location of protein bands, completeness of elution and proteinconcentration in eluted fractions are determined by spectroscopy at 280III/1., or by staining of indicator sections.

In gel electrophoresis, beef liver orgotein is found betweenslow-moving, gamma globulin protein type fractions and the fast-moving,albumin-type protein fractions.

Another preferred means for removing albumin and other types ofextraneous proteins remaining after the previously describedfractionation steps is by chromatography, e.g., using aschromatographing media porous resins which filter proteins according tomolecular volume, i.e., act as molecular sieves. One such resin isSephadex (Pharmacia, Upsala, Sweden) a cross-linked dextran resin ofdefined pore size. The partially purified orgotein in a buffer-Me++solution, is deposited in highly concentrated form on a column of theresin and then eluted in the manner coventional for chromatographiccolumns, but using a butter solution containing a divalent metal ofionic 10 radius of 0.60 to 1.00 A., preferably 0.65 to 0.79 A., e.g.,magnesium, or a mixture of two or more of magnesium, copper and zinc, aseluting solvent. Ionic strength variations often facilitate separationand subsequent elution.

In US. 3,579,495, there is disclosed a process for isolating orgoteinfrom red blood cells. According to that process, the red cells areseparated from the plasma of the blood by centrifuging. Repeated washingof the separated cells with isotonic solvents and re-centrifugingremoves residual plasma and with it the plasma albumin, adhering to thecompacted cells. The plasma-free red cells are then ruptured byhemolysis, using conventional procedures. See M. Moskowitz and M.Calvin, Exp. Cell Res., 3, 33 (1952); S. S. Bernstein et al., J. Biol.Chem., 122, 507 (1938). Hemolysis with deionized water and sonificationat 0-5 C. is preferred.

The hemoglobin and stroma are separated from the lysed mixture bymethods known in the art. See E. R. Waygood, Methods in Enzymology, Vol.2, 836 (1955), Academic Press. Preferably, for hemoglobin this is accomplished by adding a halogenated aliphatic solvent which apparentlyform an insoluble complex with the hemoglobin, along with awater-miscible organic solvent to bring a small proportion of theimmiscible solvent into the aqueous phase. Hemoglobin complex and stromathen can be removed by centrifugation.

The supernatant, now substantially free of hemoglobin and stroma, isthen freed of carbonic anhydrase and other enzymes by heating thesupernatant in the manner described in US. 3,579,495 and Example 4herein, until the carbonic anhydrase has been inactivated by heating,i.e., 10-30 minutes at 60-70 C. Thereafter the mixture is immediatelycooled to well below room temperature. The precipitated proteins areremoved by filtration or centrifugation. The supernatant remaining afterremoval of the precipitated proteins contains the orgotein protein asthe, or one of the, predominate proteins. After removal of theprecipitate formed in the heating step, the orgotein protein in theresulting solution, or isolated therefrom by dialysis andlyophilization, can be purified and isolated by mixed bed resinfiltration, electrophoresis and/or gel filtration through a polymerwhich acts as a molecular sieve, as described herein.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

EXAMPLE 1 The following is a general procedure for isolating proteinsfrom natural sources thereof to provide a suitable startingproteinaceous material for the process of this invention.

Mechanically remove as much extraneous material as possible from afreshly harvested, washed and cleaned plant or animal source of protein.In the case of animal tissue, glands and organs, remove fat, connectivetissue and blood vessels. Conduct all subsequent steps below 5 C.,except as indicated.

(a) Toluene Method Homogenize the protein source and immediately add 3vol. of deionized water or a suitable buffer, 0.05-0.30 M, e.g.,maleate, phosphate, tris -maleate, barbital,trishydroxymethyl-aminomethane, borate, cacodylate, glycine-sodiumhydroxide, etc., containing 1X10 to 2 10 M of a water soluble salt,e.g., chloride, sulfate, phosphate, acetate, citrate, maleate, borate orphosphate, etc., of a physiologically essentially divalent metal, e.g.,calcium, cobalt, copper, iron, magnesium, manganese or zinc. Adjust topH 7.0-7.8. Stir the resultingmixture for several hours. Then add slowly0.01 VOL-equivalent of toluene and continue stirring for several morehours. Let sit until the supernatant is reasonably clear. Filter, e.g.,

through cloth, cotton, glass wool or filter-aid, or cntrifuge. Excludedirect light in these operations. Immediately freeze the filtrate andlyophilize it. If direct lyophilization proves difficult, dialyze firstagainst 0.001 M buffer containing 0.1--5 10 M bivalent metal salt, e.g.,Ca++, Co++, Cu++, Fe++, Mg++, Mn++, Zn++. The resulting powder can bestored in the cold, preferably at below C.

(b) Acetone Powder Method Suspend finely disintegrated whole tissue inany of the bu1lFer--Me++ mixtures of (a) above, bring to pH 7.0- 7.8 andcool the dispersion to 0. Add to the dispersion very slowly 10 vols. ofacetone at 10 C. with rapid mechanical stirring. Let settle for about 10minutes, and decant the supernatant aqueous acetone. Collect theprecipitate either by centrifuging or by vacuum filtration through a No.l Whatman paper on a wide Buchner funnel in a cold room at 0. Wash theprecipitate twice by suspending on each occasion in about 3 vol.(Calculated from the original volume of dispersion) of acetone at 10 C.Remove the acetone from the precipitate, first using a stream ofnitrogen followed by drying the powder in vacuo over H 80 The lastacetone treatment can be followed by washing with dry peroxide-freediethyl ether (at 15), which greatly facilitates rapid drying. Store thedried material in the cold, preferably in vacuo over a drying agent.

Alternatively, disintegrate the whole tissue directly in 10 vol. ofacetone at -15 in a Waring Blendor (for 3 minutes), and retreat theprecipitate with acetone as described above.

If the first acetone precipitate contains much lipid mate rial, washingit with n-butanol at 15 greatly improves the subsequent extractions.

Alternatively, cut 1 kg. of fresh bovine liver, free from connectivetissue, into five or six pieces, rinse with tap water and mince.Homogenize portions of mince (200 g.) in a Waring Blendor with 200 ml.cold iso-osmotic KCl solution for 20 sec. Immediately mix the homogenatein the blendor with 200 ml. of acetone at 10 for another 20 sec. Pourthe acetone-treated hemogenate with stirring into a 10 liter beakercontaining 2.5 liters of acetone at 10. When the final portion of mincehas been treated,

add to the contents of the beaker cold acetone to a volume of 10 litersand mix. Hold at 4 for a few minutes. Decant the clear supernatant andagain mix the contents of the beaker with acetone to 10 liters. Decantthe clear supernatant and filter the suspension rapidly on a Buchnerfunnel covered with a sheet to exclude as much air as possible. Beforethe cake on the funnel is completely dry, wash with 2 liters of coldacetone.

Continue the filtration until the particles are completely dry. Break upthe solid material, spread out on filter paper and air-dry, preferablyunder a cover of nitrogen. Finely grind the powder while cold and storein vacuo at 4. The yield is about 250 g. of powder.

EXAMPLE 2 The following is a general procedure for producing andisolating orgotein from protein sources of the type produced in theabove-described Preparation 1.

All operations are carried out in 0.1 M tris-maleate- Me++ buffer at pH7.4, unless otherwise indicated. 0.05 M to 0.2 M tris-phosphate-Me++,tris-succinate-Me++, trisglycine-Me++ and tris-I-ICl-Me++ buffers workequally well. All operations involving organic solvents are carried outat 0 to 2 C., or lower using organic solvents precooled to 10 C. Allother operations are at temperatures below +5 C., except as indicated.

A. Removal of Buffer-Insoluble Material In the cold and in the absenceof direct light, stir 100 g. of dry powder, obtained according toPreparation 1, into one liter of tris-maleate buffer. After severalminutes add 6.5 g. MgSO 7H O in portions and adjust pH to 7.4 with INsodium hydroxide. Then add an additional 600 ml. of tris-maleate bufferand an additional 6.5 g. MgSO 7H O. Re-adjust to pH 7.4. Then add anadditional 400 ml. water and continue stirring in cold room until about6 hours have elapsed from the start of the operation. Let the mixturesettle and then filter or centrifuge. Adjust the filtrate to pH 7.8,hold in the cold until precipitation is complete, centrifuge and filtersupernatant. For storage, lyophilize the filtrate as described in thePreparation.

With some raw materials, e.g., liver, the above step and the antecedentPreparation 1 preferably is carried outwith 0.1 M manganese sulfateproviding the bivalent metal. Transchelation, i.e., removal of mostmanganese and replacement by magnesium, is achieved usingtris-maleatemagnesium salt buffer in a subsequent step. In some cases,it is desirable to carry the manganese-tris-maleate buffer through thepigment removal step and sometimes even through the heat-treatment step.In still other cases, use of buffer containing other bivalent ions,e.g., Ca, Co, Cu, Fe, Zn, may be desirable in the initial and/orintermediate steps. About a 50% or less yield of powder is obtained,based on the dry weight of the proteins in the This step often isnecessary with protein fractions obtained from dark-colored sources,e.g., liver, kidney, lung, spleen, 'jackbeans, certain bacteria, etc.Proteins from other tissues, such as testes, pancreas, placenta withblood clot removed, thymus, heart and other muscle, marine animals andother microorganisms usually do not require this step.

g. of powder from Step A are suspended with stirring in 400 ml. cold 0.1M tris-maleate-Mg++ buffer at pH 7.2. Let stand in cold room until theprecipitate has settled, centrifuge at l2 C. and decant.

If pyridine is used, divide the decanted liquid into five equal parts.Slowly and with stirring, add to each 8.0 ml. of pre-cooled c.p.pyridine. Then add 40 ml. of 0.1 M tris-maleate-Mg++ buffer to eachportion. Centrifuge at 12 C., e.g., at 13,000 r.p.m. for 15 minutes.Decant and recombine the supernatants. Discard the precipitate.

If chloroform-ethanol is used, to the decanted liquid slowly and withstirring add 15% by volume of a precooled mixture of one part chloroformand two parts ethanol. The temperature should remain below 4 C. duringthe addition. Centrifuge, e.g., at 12l4,000 r.p.m. for about 10 minutes,and discard the dark-co1ored precipitate. The lightly colored,opalescent supernatant is kept cold.

At this point, the partially purified desired protein can beprecipitated 'with solvent, the precipitate freed of adhering solvent invacuum, re-dissolved in about 0.15 M tris-maleate-Mg++ buffer, pH 7.4.Any insolubles are removed by centrifugation and discarded.

The thus-obtained solution can be lyophilized, with or without priordialysis. The resulting off-white powder is stable for several monthswhen kept in the freezer.

C. Less Soluble Material Removal The cold tris-maleate-Mg++ buffersolution from Step A or B at a pH of about 6.0 to 7.5, or its freshlymade equivalent from lyophilized powder, is brought to 40- 45% ofsaturation with ammonium sulfate, added with stirring in portions eitheras solid or as saturated aqueous solution. Hold the temperature at 05 C.Keep the mixture 10-30 minutes in the cold until precipitation iscomplete. Centrifuge at 8,00012,000 r.p.m. and discard the precipitate.Keep the supernatant for several hours in the cold. Centrifuge anddiscard any further precipitate.

Alternatively, to a 250 ml. portion of the supernatant obtained fromStep B or 62 g. of pigment-free protein obtained in Step A and dissolvedin 250 ml. of 0.1 M trismaleate-Mg++ buffer at pH 7.5, slowly add withstirring about 0.9 volumes of ethanol or about 0.75 volumes of acetonepre-cooled to 10 C., i.e., an amount suflicient 13 to precipitate only aportion of the proteins. The temperature of the mixture should notexceed +2 C. As soon as precipitation is complete, centrifuge in thecold, e.g., at 8,000-12,000 r.p.m. and discard the precipitate.

D. Heat Labile Protein Removal Heat the supernatant obtained from Step Cin a roundbottom flask or glass lined kettle in a bath kept at 65- 70 C.Stir the solution vigorously until the temperature of the contentsreaches about 59 C. and hold for about 20 minutes at or near thistemperature. Immediately thereafter immerse the flask into a DryIce-solvent or an icesalt bath and continue stirring vigorously untilthe temperature has dropped to 25 C. Centrifuge the resulting bulkyprecipitate in the cold and discard.

E. More Soluble Material Removal (1) Salt Precipitation: Preferably at apH of about 6.0 saturate the buffer solution from Step D with ammoniumsulfate as above to 58-76% and hold /2 to 2 hours in the cold untilprecipitation is complete.

Take up the nearly White ammonium sulfate precipitated material in 10-15times its weight of 0.10 M trismaleate-Mg++ buffer and hold for /2 hourin the cold. Centrifuge off and discard any insoluble or furtherprecipitate.

(2) Solvent Precipitation: To the same starting solution as used in (1),preferably at a pH of about 3.5 or about 7.5, add in portions and withstirring pro-cooled (10 C.) acetone or ethanol, keeping the temperaturearound C., in an amount suflicient to precipitate at least a portion ofthe proteins in solution. If, for instance, 0.5 volumes of acetone wereused to precipitate less soluble proteins, use about 0.5 to 1.5additional volumes. Keep the mixture for a few minutes at about 0 C.until the precipitation is complete. Centrifuge the precipitate at 1 C.or below (10,000-13,000 r.p.m.). Separate and free the precipitate fromadhering solvent under vacuum in the cold. Check the supernatant for theextent of precipitation by addition of 0.2 volume of pre-cooled C.)acetone or ethanol. Isopropyl alcohol (0.5-2 v./v.) can also be used.

Preferably, ammonium sulfate fractionation is used to precipitate theless soluble proteins and solvent fractionation to precipitate thedesired protein.

F. Electrophoresis Fractionate a solution of the protein precipitated byStep E in 10l5 times its Weight of 0.1 M tris-maleate- Mg++ buffer(discarding any insoluble material) by gel electrophoresis usingpolyacrylamide with the running gel at pH 8.0 to 9.5 or at pH 3.0 to4.3.

The following apparatus is used in the gel electrophoresis step:

Electrophoresis Chamber; Buchler Power Supply; Loading Rack; Syringes(disposable plastic 20 cc., 5 cc., 1 cc.); Syringe Needles 22G x 1 /2and 25G x Teflon Tips for layering tool; Pho-topolymerizing light(fluorescent light source).

The electrophoresis chamber should be thoroughly washed and rinsed indistilled water. The chamber is then immersed in a Siliclad (Clay-Adams,New York) solution (1 part Siliclad to 50 parts of water) for a fewminutes. It is then rinsed and oven-dried.

The electrophoresis chamber used is the special production modeldescribed heretofore.

The reagents used are the following:

Acrylamide monomer (Eastman No. 5521); N,N'-Methylenebisacrylamide BIS(Eastman No. 8383); Riboflavin (Eastman No. 5181); N,N,N,N'Tetramethylethylenediamine TEMED (Eastman No. 8178); Glycine (EastmanNo. 445); TRIS (T ris-hydroxymethyl-aminomethane) (Fisher T-395);Sucrose (Baker No. 4072); Ammonium persulfate Reagent grade; 1 N HCl; 1M H PO Acetic Acid; and Methanol (Reagent Grade). Small pore gel, 7% isused.

The stock solutions used are:

(a) 1 N HCl, 480 ml.; TRIS, 363 g.; TEMED, 4.6 ml.; and H 0 to make1,000 ml. (b) 1 M H PO 256 ml; TRIS, 57 g.; and H 0 to make 1,000 ml.(pH 6.9). (c) Acrylamide, 280 g.; BIS, 7.36 g.; and H 0 to make 1,000ml. (d) Acrylamide, g.; BIS, 25 g.; and H 0 to make 1,000 ml. (e)Riboflavin, 40 mg. and H 0 to make 1,000 ml.

Working solutions used are:

Mixture A: 1 part (a); 2 parts (c); and 1 part (H O). Mixture B:Ammonium persulfate, 1.00 g.; and H 0,

The buffer solution used is: TRIS, 60 g.; Glycine, 288 g.; H O to 20liters; pH, 8.45.

Tracking Dye used is 0.001% Bromphenol Blue. It was found that for thebest separations fresh buffer should be used for each run. The buflercan be used for a maximum of 3 runs with some loss of resolution on thesecond and third runs. All solutions should be stored in therefrigerator. If they are, they are usable for several months, exceptMixture B which should be made fresh weekly.

Equal parts of Mixture A and Mixture B taken directly from therefrigerator are mixed in a filtering flask. The flask is attached tothe aspirator and the contents are swirled gently for about a minute ina partial vacuum. The chamber is then filled with the cover on, to inchfrom the top using a long thin tube. The thin tube is inserted to thebottom of the chamber and is slowly withdrawn as the slab is filled,keeping the tip beneath the gel surface. The gel is then water layeredand placed on top of a drying oven. Gelling is complete in about 15 to30 minutes.

A water layering tool can be made from a plastic syringe and a 25G x /8inch needle tipped with a Teflon tip (Analytical Chemists, Inc.). Thesyringe is filled about /3 full with Water, tinted blue with trackingdye solution. The Teflon tip is placed just beneath the surface of thegel and moved upwards as the water is expelled onto the gel. The tipshould at no time be lifted above the surface of the liquid.

After polymerization, the water is carefully removed. The surface isrinsed once with degassed sucrose gel.

The sample (0.5-5.0 g.) is suspended in water, 0.9% saline or in TRISbuffer, mixed with gel and filled into the pre-formed trough of the gelslab. The surface is sucrose layered and sealed with a capping gel.Capping gel is added until a convex miniscus is formed. A plastic coveris then slid across the top so that no air bubbles are trapped. Theloaded trough is placed between two fluorescent lamps as close to thelights as possible. Polymerization is complete between 30 minutes and anhour depending on the amount and nature of sample used.

When polymerization is complete as indicated by opacity of sample andcapping gels, the cover is removed and the chamber loaded into thebuffer reservoirs. The apparatus and buffer are pre-cooled before a runand the run is made at about 5 C. or less. The power supply is set togive constant current which is set at 100-200 ma. depending on theamount of sample and the size of the gel slab. A run takes 2-6 hours andat the end the tracking dye will have traversed nearly the whole slab.

After the run, the desired orgotein is found in the area comprisingabout 20-30% of the distance travelled by the tracking dye from thepoint of origin. It is well-separated from the much faster travellingalbumin and albumin-type fractions and also well-separated from thesmall amounts of much slower travelling extraneous protein fractions.

The desired orgotein is eluted from the gel by a crosscurrent oftris-maleate buffer 0.1 M containing 0.001 M Mg++. Progress of theelution is monitored by U.V. absorption at 280 m Uniformity is checkedby analytical disc gel electrophoresis followed by staining with AmidoBlack. If desired, the albumin-type and the slow frac- 15 tions can berecovered in a similar manner using trismaleate buffer.

In the cationic system at pH 3.8 in the running gel, potassium ion isused as the leading ion and fl-alanine as the trailing ion. Acetic acidis used as the buffer. The procedure followed with these systems is thesame as that used with the anionic system (running gel pH 9.4).

To isolate the desired orgotein from the eluate, dialyze exhaustivelyagainst 0.001 M tris-maleate-Mg++ buffer and then against deionizedwater and lyophilize. A white flutfy powder representing about 616% ofthe ammonium sulfate or solvent precipitated product is Obtained.

A typical overall yield of isolated orgotein is 0.005 to 0.015%,calculated on the dry weight of the original source material.

EXAMPLE 3 All operations, unless otherwise indicated, are carried out ina cold room (2-5 C.).

Preparation Fresh beef liver is ground into a plastic container. Colddistilled water (two liters per kg. of liver) is added with stirring andthe mixture is adjusted with 0.1 N sodium hydroxide to pH 7.5 to 7.6.Sufficient 2 M manganese sulfate solution is added to bring the molarityof the mixture to 0.05. The pH is adjusted to 7.6 and fresh cold Wateris added to bring the water to three liters per kg. of liver.Thereafter, 50 ml. of toluene per kg. of liver are added and the mixtureis stirred in the cold room overnight.

Removal of More Soluble Material The next morning the suspension ispassed through plastic gauze and to the filtrate 1.5 volume of coldacetone (-10 C.) is added with gentle stirring. The acetone is addedthrough a glass tube extending well below the surface of the mixture.The ensuing precipitate is immediately collected by centrifuging andthen right away suspended with about 25% (v./v.) of 0.05 M maleate-Mnbuffer, calculated upon the volume of the filtrate before addition ofthe acetone. The mixture is stirred in the cold room for several hours,passed through plastic gauze and clarified by centrifuging.

Heat Labile Protein Removal The supernatant is heated rapidly to about60 C. with stirring in a stainless steel or glass lined kettle andmaintained at or close to 60 C. for about 20 minutes. Thereafter, themixture is cooled to about 5 C. as rapidly as possible and the bulkyprecipitate is removed in the cold room by slow suction over a broadfilter surface or by centrifugation. The clear solution is brought to 25C. and 0.9 volume of denatured ethanol C.) is added from a reservoirthrough a glass tube extending well below the surface of the mixture.Effective stirring is essential and the temperature must remain at 5 C.or lower.

After the addition of the alcohol has been completed, the mixture iskept in the cold room just long enough to permit the precipitate tocompact and to settle. The precipitate is recovered by vacuum filtrationor centrifugation and immediately dissolved in cold 0.001 M maleate-Mn++ buffer, pH 7.0. The amount of buffer is approximately 4 v./wt. Thesolution is clarified by centrifuging, the supernatant decanted, theprecipitate re-extracted using small amounts of cold buffer, thesupernatants combined and lyophilized. Prior dialysis to remove butferions, while possible, is not necessary at this point. The resultantpowder is stable for several months at room temperature butpreferentially is kept in the cold room. It represents a mixture of thedesired orgotein, arginase and other enzymes, albumin and othernon-essential proteins.

Removal of Less Soluble Material and Transchelation For furtherprocessing, this powder is dissolved in about 12 times the volume ofcold 0.2 M Tris-0.001

M :Mg bufier, pH 7.8. This solution is treated with cold saturatedammonium sulfate solution, 0.001 M in Mg++. Five increments of 375 ml.each are added per 1000 ml. of buffer solution. The respective states ofsaturation achieved by this technique are 15%, 30%, 45%, 60% and 75%. Ineach instance the addition of the ammonium sulfate solution is carriedout slowly at 0-5 C. with stirring. Stirring is continued for anotherten minutes and the resulting precipitate is collected by centrifugingat 4500 r.p.m.s for thirty minutes at 0 C.

Of the five precipitates obtained, the first one (A) is discarded. Itrepresents high-molecular Weight-protein impurities. The second andthird precipitates (B and C) are combined. They represent arginase andother enzymes which can be processed separately for the isolation ofthese products. The fourth and fifth (D and E) are also combined. Theycontain the desired orgotein, in a still crude state, contaminated withalbumin and various other proteins both of lower and higher molecularweight. The final supernatant is discarded. It contains low molecularproteins and other undesirable impurities.

Chromotography Precipitates D and E are dissolved in 0.03 MTrisglycine0.001 M Mg++ buffer, pH 7.8 at a concentration as close to10% (w./v.) as possible and dialyzed against cold buffer until negativeto sulfate ion. The dialyzed solution is clarified by centrifugation andthe supernatant is passed through a Millipore filter. The filtrate isapplied directly to the head of a chromatography column (3 x 18 inches)filled with Sephadex G- (crosslinked dextran resin, Pharmacia, Sweden).The Sephadex has been swelled, defined and washed by standard techniquesdescribed in literature of the manufacturer. The packed column isequilibrated with 0.03 M Tris-glycine0.001 M Mg++ buffer, pH 7.8 andadjusted to a flow rate of about 20 ml. per hour.

After application to the column, the sample is permitted to equilibratewithin the first few cm. of the resin bed for approximately 30-45minutes when fractionation is started. Individual fractions of up to 10ml, are collected. The emergence of peaks is determined by measuring theprotein concentration by the absorbance at 280 millimicron.

Two and sometimes three peaks emerge from the column prior to theemergence of the desired orgotein. They represent albumin and otherundesirable protein impurities of similar or larger molecular weight.Fractions representing these peaks are discarded. The desired proteingenerally emerges in the range of 100-150 ml. of total eluate. Thesefractions are combined for further processing. Residual, lower molecularweight protein impurities emerge from the column on further elution,particularly on increasing the ionic strength of the bulfer. They areremoved to clear the column for a subsequent run.

The combined fractions containing the desired orgotein Butter and ExcessMg++ Ion Removal are dialyzed against deionized H O-0.001 M Mg++ untilthey contain less than 10 M Tris buffer. Thereafter, dialysis iscontinued against deionized water containing 1- 5 10 Mortho-phenanthroline or ethylenediamine tetraacetic acid salts until theconcentration of Mg++ has been reduced to less than 10* M. If a proteinchelate is desired whose predominant metal is other than magnesium,e.g., calcium, copper, iron or zinc, expose the protein after dialysisfor about a day to a soluble salt of the metal of choice of a molaritywhich maintains the protein in solution, and then remove excess metalion in the manner described above. The resultant solution is clarifiedby centrifuging and the supernatant is treated in either of two ways.For the preparation of bulk protein powder the solution is lyophilized.For the preparation of sterile protein solution for injection purposes,dextrose is added to the solution to 5% w./v. The dextrose solution isthen sterilized by Millipore filtration and filtered into pre-sterilizedampules or vials under sterile conditions to be used as such or as alyophilized powder.

75 kg. of fresh beef liver, which contains about 70% water and isequivalent to about 22.5 kg. of dry matter, yields about 200 grams (1%)of the Mn++ chelate intermediate.

200 Grams of the Mn++ chelate yield 12.5-17.5 grams (0.060.08%) ofcombined D and E fractions. On Sephadex chromatography, these amounts ofD and E fractions yield 2.4 to 2.9 grams of the desired protein,equivalent to an overall yield of 0.0l1-0.014% calculated on the dryweight of the liver.

EXAMPLE 4 The following are examples of an improved process for theisolation of orgotein claimed in the application of W. Huber, Ser. No.150,809 now US. 3,687,927, filed June 7, 1971, as a continuation-in-partof Ser. No. 657,866, filed August 2, 1967, now abandoned.

All operations, unless otherwise indicated, are carried out in a coldroom (2-5 C.).

a. Removal of Insoluble Material Finely macerated fresh beef liver ismixed with cold 0.025 M tris-glycine buffer containing 0.01 M Mn++ at pH7.5 (two liters per kg. of liver). Adjust pH to 7.5 if necessary.Thereafter, if the liver is fatty, 50 ml. of toluene per kg. of liverare added. The mixture is stirred 4-6 hours. The resulting suspension iscentrifuged at 20,000 G for 10-20 minutes or pressed through plasticgaugze and the insolubles discarded.

b. Removal of More Soluble Material To the aqueous filtrate obtained inthe preceding step is added rapidly and with thorough agitation 1.25volumes of cold acetone (10 C.) through a glass tube extending wellbelow the surface of the mixture. The ensuing precipitate is immediatelycollected by centrifuging, e.g., for

10 minutes at 20,000 G. Completeness of precipitation is checked byadding an additional 0.25 to 0.50 volumes of acetone to the filtrate.Any additional precipitate is also collected. The precipitated proteinsare quickly suspended with about 25% (v./v.) of 0.025 M tris-glycinebuffer at pH 7.5 containing 0.01 M Mn++, calculated on the volume of thefiltrate before addition of the acetone. The mixture is stirred in thecold room for several hours. The insolubles are removed by centrifugingand the clear supernatant is adjusted to achieve an about 10% proteinconcentration. Protein concentration can be determined by Biuretanalysis or other standard method.

c. Heat Labile Protein Removal The thus-obtained buffer solution isheated rapidly to about 60 C. with stirring in a stainless steel orglass lined kettle and maintained at or close to 60 C. for about 20minutes. Thereafter, the mixture is cooled to about 5 C. as rapidly aspossible and the bulky precipitate is filtered in the cold room by slowsuction over a broad filter surface or centrifuged at 12,000 to 16,000 Gfor minutes. The precipitate is re-extracted, using small amounts ofcold buffer, and the clear supernatants combined. The precipitate isdiscarded.

Removal of Less Soluble Material and Transchelation The solution fromthe heat treatment step is concentrated, if necessary, to a proteinconcentration of about 8%, e.g., using an ion selective membrance(Diafio Membrane, Amicon Corp., Cambridge, Mass.) to remove excessbuffer. The protein solution is mixed slowly and with stirring with coldsaturated ammonium sulfate solution containing 10 M Mg, 10 M Cu++ and 10M Zn to a 40% (NH SO concentration. Stirring is continued for anotherminutes and resulting precipitate is removed by centrifuging at 20,000 Gfor thirty minutes at 0 C. and discarded. To the filtrate is added 18 anadditional amount of the saturated ammonium sulfate solution to bringthe protein solution to 65% (NH SO concentration. The resultingprecipitate contains the desired protein and is collected bycentrifugation or filtration. The final supernatant is discarded.

e. Gel Filtration The final precipitate from the (NH SO step isdissolved in 0.025 M tris-HCl or tris-glycine or 0.01 M phosphate orborate buffer, containing 10- M Mg, 10-* M Cu and 10- M Zn++, at pH 7.8to a concentration as close to 10% (w./v.) as possible and dialyzedagainst cold buffer until negative to sulfate ion. The dialyzedsolution, after clarification by centrifugation, if necessary, is passedthrough a Millipore filter. The filtrate is applied directly to the headof chromatography columns (3 X 18 in.) filled with Sephadex G-l00 orG-75 (epichlorohydrin cross-linked dextran resin, Pharmacia, Sweden).The Sephadex has been swelled, refined and washed by standard techniquesdescribed in literature of the manufacturer. The packed columns areequilibrated with one of the above-described buffers and adjusted to aflow rate of about 20 ml. per hour. The addition of 5-10% dextrose orsucrose to the solution improves uniformity of adsorption, whichfacilitates subsequent resolution.

After application to the column, the sample is permitted to equilibratewithin the first few cm. of the resin bed for approximately 30-45minutes, at which time fractionation is started, the column beingdeveloped with additional buffer solution. Individual fractions of up to10 ml. are collected. The emergence of peaks is determined by measuringthe protein concentration by the absorbance at 280 millimicron.

Two and sometimes three peaks emerge from the column prior to theemergence of the desired protein. They represent albumin and otherundesirable protein impurities of similar or larger molecular volume.(Fractions representing these peaks are discarded. The desired proteingenerally emerges in the range of -170 ml. of total eluate. Thesefractions are combined for further processing. Residual, lower molecularweight protein impurities emerge from the column on further elution,particularly on increasing the ionic strength of the buffer. They areremoved to clear the column for a subsequent run.

f. Buffer and Excess Me Ion Removal The combined fractions containingthe desired protein are filtered through a column of mixed bed resinA-mberlite MB-l Monobed gel-type Ion Exchange Resin, (Rohm & Haas), astyrene-divinyl benzene strongly acidic (-SO -H+) strongly basic columnis then back-washed several times with deminer-- alized water toconstant pH (ca. 7.0) and ionic strength (conductance about 1.0 mho) ofthe effluent. The final' bed height is 33 inches, giving a bed volume of58.3 cubic' inches (957 milliliters) and total exchange capacity of 440mini-equivalents, based on a factor of 0.46 given by the manufacturerfor this resin.

The fractions from the gel filtration step containing the desiredprotein are combined and concentrated, if

necessary, to a protein content of 810%. This solution is carefullyloaded onto the top of the column and thereafter developed withdemineralized water. The flow rate is adjusted to about 20 millilitersper minute and the appearance of the protein in the eluate is monitoredby ultraviolet absorption (A The eluate is collected in about 25milliliter fractions. The desired protein generally appears in thefourth to twelfth fractions. Buffer- Me concentration drops well below10 M, as indicated by a drop of conductivity from 4,000 to 5,000 mhobefore column filtration to 1.5 2.5 mho thereafter.

For the preparation of a sterile protein solution for injectionpurposes, fructose, sucrose or other saccharide is added to theresulting buffer solution to a concentration of 2 parts saccharide perpart protein. The solution is then sterilized by ultra-filtration andfiltered into presterilized a-mpoules or vials under sterile conditions.The resulting product can then be lyophilized to produce a more stableproduct.

Following the above-described process, 75 kg, of fresh beef liver (22.5kg, dry weight), yields about 2540 grams (0.12-0.17) of finalprecipitate from the step and 7 to 9 grams of the final, fully purifieddesired protein, equivalent to an overall yield of 0.032 0.041%calculated on the dry weight of the liver, a 300 or more percentincrease in yield over that obtained by the process described in Ser.No. 576,454.

' In an alternative procedure, the filtrate from Step (a), instead ofbeing diluted with acetone, is first heated for about 20 minutes at orclose to 60 C., then rapidly cooled to about C. The resultingprecipitate is removed by filtration or centrifugation and discarded.The filtrate is then treated with acetone as in Step (b). Step (c) isomitted. The precipitate obtained from the ace tone treatment isdissolved in 0.025 M tris-glycine buffer containing M Mg++, 10 M Cu++,10 M Zn++ at pH 7.5 adjusted if necessary to a protein concentration ofabout 8% and then treated as in Steps ((1), (e) and EXAMPLE 5 Thefollowing is an example of a process for the isolation of orgotein fromred blood cells claimed in the application of W. Huber, Ser. No. 815,175filed Apr. 10, 1969, now US. 3,579,495.

Fresh beef blood was centrifuged at 2,600 G for 10 minutes at 0 C. andthe plasma decanted. The red cells were then washed repeatedly with 2 to3 volumes of 0.9% saline solution. The washed red cells were hemolyzedby mixing with 1.1 volumes of cold deionized water containing 0.02%detergent (Saponin). After a minimum of 30 minutes at 4 C., 0.25 volume(per volume of hemolysate) of ethyl alcohol at C. was slowly added withstirring followed by 0.31 volume (per volume of hemolysate) ofchloroform, also at 15 C. Stirring was continued for about 15 minutes at'-5 C. or below, at which time the mixture was a thick paste. Thehemoglobin precipitation was carried out in a cold bath which was keptat below 10 C. After the paste had stood for a further 15 minutes at 4C., 0.2 volume of cold 0.15 M NaCl solution was added, giving an easilypoured suspension. The precipitate and excess chloroform were removed bycentrifuging at 20,000X G at about 10 C. for 10 minutes. The supernatantliquid was filtered and dialyzed against cold-deionized water. Thedialyzed solution was lyophilized.

The alcohol-chloroform precipitate was re-extracted with a minimumamount of deionized water by blending the precipitate and water in ablender and centrifuging. Usually, a volume of water equal to that ofthe volume of starting red blood cell is needed. The re-extractionsolution was dialyzed and lyophilized. Re-extraction of the precipitatedhemoglobin often yields 30-50% of protein mixture present in theoriginal supernatant. Depending upon the structure of the precipitate, asecond reextraction may give an additional 10-15%.

The lyophilized material Was re-dissolved in 0.025 M tris-glycine buffercontaining 0.001 M Mn++ at pH 7.5 (usually to a concentration ofmg./ml.). The solution was heated at or close to 65 C. for about 15minutes. This step removes carbonic anhydrase and other heat labileenzymes from the solution. After heating, the solution was quicklycooled in an ice bath to about 5 C. The solution was then centrifuged at20,000 G at 0 C. for 10 minutes to remove the precipitate. Thesupernatant was dialyzed against deionized water to remove excess metalions and buffer and then lyophilized. The resulting solid is rich inorgotein.

(a) Gel Filtration Sephadex G-75 is slowly added to warm deionized water(approximately 60 C.) with continuous stirring. The vessel containingthe mixture is then placed in a 60 C. water bath for five hours and 45minutes, removed and allowed to stand for one hour at room temperature.The supernatant and fines are decanted by suction. Buffer is added tothe swollen Sephadex gel at four to five times its volume. The Sephadexgel is stirred, allowed to settle, and the fines and supernatant removedby suction. Fresh buffer is again added to the swollen gel, and theabove process repeated four times. The final suspension is chilled to 4C. and then deaerated under reduced pressure before use.

A recirculating column made of polymethacrylate is used. The column is1050 mm. long and has an internal diameter of 32 mm. In filling thecolumn with degassed buffer, special care is taken to insure that no airbubbles are trapped near the filter and on the sides of the column. Thebuffer filled column is then moved into the cold room and clamped into avertical position with the aid of a carpenters level. Afterequilibration in the cold room, the gel slurry is poured into a funnelconnected to the top of the column with continued mechanical stirring.When a layer of Sephadex a few centimeters thick has formed on thebottom of the column, the outlet at the bottom of the column is openedto allow an even flow. During the packing, a rising horizontal surfaceof gel in the tube indicates proper uniformity in packing. Afterapproximately cm. of gel has settled, the excess gel and buffer areremoved. After the top surface of the gel has completely settled, thetop of the column is closed with a plunger fitted with a filter disc.Buffer is then circulated through the column for two days in order tostabilize the bed. Flow rate is maintained at 10 ml. per hour. Final bedvolume V=1rr h=(3.l4) (1. 6 cm.) (96.5 cm.)=775.7 cc.

The lyophilizate from the heating step is dissolved in buffer (20mg./ml.). Insolubles, if present, are removed by centrifugation followedby Millipore filtrations. The clear solution is loaded on the columnusing an LKB selector valve (Model 4911B) All column runs are performedat 4 C. The buffer used is 0.05 M Tris-HCl, pH 7.5, 0.15 M in KC] and0.005 M in glycine, containing 10 M Mg++, 10- M Cu++ and 10- M Zn++.

The protein solution is loaded from the bottom. Ascending buffer flowrate is maintained at 10 ml. per hour. Protein content of fractions isdetermined by absorbance at 280 m,u.

The elution volume for each protein can be monitored both volumetricallyand gravimetrically.

"If prior processing has proceeded normally, the first peak whichemerges from the column is orgotein. It generally emerges in the rangeof 300-400 ml. of total eluate. These fractions are combined for furtherprocessing. Following the main, well defined peak is sometimes ashoulder which contains orgotein mixed with small amounts of a minorimpurity, which need not be separated. However, it can be separated bycollecting the eluate fractions separately, and then further purified byrecycling. Lower molecular weight protein impurities emerge from thecolumn substantially later, upon further elation. They are removed toclear the column for a subsequent run.

(b) Buffer and Excess Metal Ion Removal The orgotein solution obtainedfrom the gel filtration is filtered through a column of mixed bed resinAmberlite MB-l Monobed gel-type Ion Exchange Resin (Rohm & Haas), astyrene-divinyl benzene strongly acidic 3* strongly basicgroup-containing mixed copolymer which reduces buffer and unbound Mg++,Cu++ and Zn++ ion concentrations to less than 10- M. Alternatively, thiscan be done by dialysis.

A column 1.45 x 45 inches is half filled with demineralized Water fromwhich all air bubbles have been removed. A slurry of the resin inair-free demineralized water is poured gently into the column andallowed to settle. The column is then back-washed several times withdemineralized water to constant pH (ca. 7.0) and ionic strength(conductance about 1.0 mho) of the efiluent. The final bed height is 33inches, giving a bed volume of 58.3 cubic inches (957 milliliters) andtotal exchange capacity of 440 milliequivalents, based on a factor of0.46 given by the manufacturer for this resin.

The eluate from the gel filtration step containing the orgotein isconcentrated, if necessary, to a protein content of 810%. This solutionis carefully loaded onto the top of the column of ion exchange resin andthereafter developed with demineralized water. The flow rate is adjustedto about '20 milliliters per minute and the appearance of the protein inthe eluate is followed by ultraviolet absorption (A The eluate iscollected in 25 to 50 milliliter fractions. The desired proteingenerally appears in the fourth to twelfth fractions. Buffer-Me++ 3concentration drops well below 10- M, as indicated by a drop ofconductivity from 4,000 to 5,000 mho before column filtration to 1.5-2.5mho thereafter.

By the same procedure orgotein congeners having the properties givenabove can be isolated in 98% purity from horse, sheep, rabbit andchicken red blood cells in 0.01, 0.005, 0.008 and 0.006% overall yield,respectively, and also from human, pig, and other mammalian red bloodcells.

EXAMPLE 6 The following is an example of a process claimed in theapplication of W. Huber, Ser. No. 3,492, filed Jan. 16, 1970, now US.3,624,251 for the removal of the small amount of tenacious extraneousprotein present in substantially pure orgotein isolated from beef liverin the manner described in Examples 1-3.

Solutions of a production lot of orgotein of about 90% purity at aconcentration of mg. protein per ml. of 0.005 M glycine buffer, pH 8.5,in 0.9% saline were pipetted into each of five clean glass containers ofequal size. This lot of orgotein contained 10.5% slow moving proteinimpurity, based on stain intensity (Amido Black) of electrophoreticallyseparated slow-moving protein. Container 1 was used as a control, andcontainers 2 to 5 were heated at 70 C. at 15, 30, 45 and 60 minutes,respectively.

After heating, the orgotein solution in each of the containers,including the control, was filtered through Millipore. A biuret proteindetermination was made on the clear filtrate. Each of the containerscontained the following:

Ml. Filtered orgotein solution 0.5 Buffer 0.5 Biuret reagent -s 1.5

The heated samples were examined electrophoretically in agarose thinfilm gels. Slow moving and backward moving material in the orgoteinsample was removed to some degree after heating 15 minutes at 70 C., and

more so after heating 30 minutes at 70 C. They were completely removedafter heating for 45 and 60 minutes at 70 C. At this point thebackground of the electropherogram looked much clearer, also. Orgoteinloss was 12-16%.

EXAMPLE 7 The following is an example of another process for the removalof the tenacious impurity present in substantially pure orgoteinisolated from beef liver in the manner described in Examples 1-3, whichprocess is claimed in the application of W. Huber Ser. No. 3,538, filedJan. 16, 1970 and now abandoned in favor of US. 3,758,682.

Impure lots of orgotein containing 28.2, 20.9 and 27.8%, respectively,of slow moving (on gel electrophoresis in thin film argarose) impuritiesand 17.1, 19.9 and 11.8%, respectively, of background impurities(causing a smearing on gel electrophoresis) were selected by virtue oftheir high content of impurities from rejected batches. In the Ungaranti-inflammatory bioassay one of these lots had failed badly while theothers had failed marginally.

The buffer used was 0.1 M phosphate, pH 6.0; NaH PO -N HPO (1:7vol./vol.). The anion exchange resin used was Whatman DEAE cellulose-52,microgranular (W. & R. Balston, Ltd., Hardstone, Kent, England). Thision exchanger is supplied wet and pre-swollen, thus obviating the needfor re-suspension.

To prepare the column. 30 g. of the DEAE-cellulose was stirred into 300ml. of 0.1 M phosphate buffer, pH 6.0. The slurry was allowed to settleand the supernatant decanted. 0.01 M phosphate buffer, pH 6.2, was addedand the mixture stirred thoroughly. The slurry was allowed to settle for10 minutes and the supernatant decanted. This step serves both toequilibrate the cellulose with the buffer and to remove the fines, whichis important since they reduce the column flow rate. Washing thecellulose with the starting buffer was repeated until both the pH andthe conductivity remained constant at the correct values. Gentle vacuumwas applied to the slurry to remove occluded air and carbondioxide. Theslurry was used immediately for column packing. If the resin is left incontact with bufiers or polyelectrolytes for longer than one week, apreservative, e.g., 0.03% toluene, should be added.

A glass column of 1.5 cm. diameter fitted with a nylon net and aMillipore filter support unit at the bottom was mounted vertically. Thecolumn was filed with 0.01 M sodium phosphate buffer, pH 6.2. Theequilibrated and relatively thick DEAE-cellulose slurry (about 120-150%of original volume) was poured into the column through a funnel attachedto the top of the column. The column top was closed until 1 cm. of thecellulose had settled at the bottom. The column top was then opened toallow free flow. A column of about 20 cm. was packed using settlingtimes of 20-30 minutes. The slow sedimenting fines at the top of thecolumn were removed by suction. The column was equilibrated by runningstarting buffer through for several hours or overnight. The pH andconductivity of the eluate were checked to ensure full equilibrationbetween the exchanger and the buffer. Flow rate was adjusted byhydrostatic pressure by placing the buffer source about 40 cm. above thehead of the column, which produces a flow rate of about 30 ml. per hourfor a column of 1.5 cm. in diameter and 20 cm. in heght with a bedvolume of 30 ml.

-200 mg. of the starting orgotein was dissolved in 2-4 ml. of startingbuffer and the resulting greenish solution layered gently over thesurface of the bed. After absorption, the orgotein solution appears as abroad greenish band near the top of the column. The column was thenconnected to the buffer reservoir and elution begun with 0.01 Mphosphate buffer, pH 6.2. Five ml. fractions were collected, using aSimplex (B. Braun, Melsungen, West Germany) fraction collector. Thecolumn was operated at room temperature and the collected fractions werecooled by ice water. Upon application of the elution buffer, abrownish-pink band separated from the sample zone on the column. Itmoved rapidly downwards and was eluted immediately after the voidvolume, requiring a buffer volume of 40-50 ml. The material had a highabsorbance at 280 my. and by subsequent gel electrophoresis was shown toconsist entirely of the slow moving impurity described above. Afterpooling of the fractions containing the slow-moving impurities, elutionwas contained with 0.01 M phosphate buffer, pH 6.2, to a total volume ofabout 300 ml. After about 120 ml. of eluate had been collected,additional material with less pronounced absorbance at 280 m, waseluted. Subsequent electrophoresis of appropriately pooled fractionsshowed this material to be composed of background impurities describedabove. After elution of the background impurities, no further materialcould be eluted with 0.01 M phosphate buffer, pH 6.2.

Elution of the orgotein was carried out by stepwise increase of bufferionic strength. No significant elution was observed until ionic strengthhad been increased about tenfold to 0.10 M, pH 6.2. At this point thezone remaining at the top of the column migrated rapidly downward as asharp, light green band with the buffer front. Complete elution wasachieved with about 60 ml. of 0.1 M buffer.

Eluted fractions 68 to 76, which contained the orgotein, were pooled,extensively dialyzed and then lyophilized. Dialysis for 3 to 5 days withnumerous changes of deionized water were required to remove allextraneous non-chelated ions. In subsequent runs, increased ionicstrength was achieved by the addition of 0.09 M NaCl to the startingbuffer. This reduced the dialysis time required to remove extraneousnon-chelated ions to about 2 days.

What is claimed is:

1. In a process for the production of orgotein which comprises at leastone step of fractionating an aqueous solution of a mixture of buffersoluble proteins comprising the orgotein, the improvement whichcomprises conducting the fractionation step with the mixture of 24proteins dissolved in a buffer solution containing dissolved therein inat least l 10 concentration at least one salt of a divalent metal havingan ionic radius of 0.60 to 1.00 A.

2. A process according to Claim 1 comprising a plurality of suchfractionating steps.

3. A process according to Claim 1 comprising a heating step in whichextraneous proteins are insolubilized.

4. A process according to Claim 1 wherein the divalent metal has anionic radius of from 0.65 to 0.79 A.

5. A process according to Claim 4 wherein the buffer solution containsdissolved therein at least one of a CW and a Zn++ salt.

6. A process according to Claim 5 wherein the buffer solution alsocontains dissolved therein both a Cu++ and a Zn++ salt.

7. A process according to Claim 4 comprising a plurality of suchfractionating steps.

8. A process according to Claim 7 comprising a heating step in whichextraneous proteins are insolubilized.

9. A process according to Claim 8 wherein the buifer solution containsdissolved therein at least one of a Cu++ and a Zn++ salt.

10. A process according to Claim 9 wherein the buffer solution alsocontains dissolved therein both a Cu++ and a Zn++ salt.

References Cited UNITED STATES PATENTS 3,579,495 5/1971 Huber 2603,624,251 11/1971 Huber 260 112 X 3,637,640 1/1972 Huber 2601 12 X3,687,927 8/1972 Huber 2601 12 X FOREIGN PATENTS 1,160,151 7/1969 GreatBritain.

HOWARD E. SCHAIN, Primary Examiner US. Cl. X.R.

